Biochip

ABSTRACT

When genetic analyses are performed using the DNA microarray of the present invention, the inspection accuracy is improved. A sample solution is supplied onto a base plate  10  to prepare the DNA microarray  20  which includes a large number of spots  80  containing capture solutions arranged on the base plate  10.  The capture solutions are adapted to specifically react with a specimen and provide information about a structure within the specimen. In the microarray  20,  the planar configuration of the spots  80  are substantially circular, and a plurality of spots having different spot sizes are formed on the base plate.

FIELD OF THE INVENTION

The present invention relates to a DNA microarray (DNA chip) whichspecifically reacts with a biochemical specimen and which is used forinspection equipment represented, for example, by a biochip to be usedin order to obtain information on a structure of the specimen,especially in which several thousand to not less than ten thousand kindsof different types of DNA fragments are aligned and fixed at a highdensity as spots on a base plate such as a microscopic glass slide.

BACKGROUND OF THE INVENTION

The method of analyzing the genetic structure has been remarkablyprogressed in recent years. A large number of genetic structuresrepresented by those of human genes have been clarified. The analysis ofthe genetic structure uses a DNA microarray (DNA chip) in which severalthousand to not less than ten thousand kinds of different types of DNAfragments are aligned and fixed as spots on a base plate such as amicroscopic glass slide.

In recent years, there is a demand for enhancing the reproducibility,the quantitative performance in the information obtained from the DNAmicroarray and obtaining much more information from the DNA microarray.The information obtained from respective spots needs to be correct,uniform, and complex.

Those widely used as the method of forming the spots for the productionof the DNA microarray are generally based on a system such as the QUILLsystem, the pin & ring system, and the spring pin system in which asample solution containing DNA fragments is supplied (stamped) onto thebase plate by using a so-called pin. When any one of the foregoingmethods is adopted, it is important to suppress the dispersion of thevolume and the shape of each spot so that the distance between therespective spots is maintained to be constant.

On the other hand, in order to realize a higher density, it is alsogreatly expected to develop a new method which is excellent inproductivity and in which the shape control performance for the spot issatisfactory.

The conventional method of forming the spot is based on the supply(stamping) of the sample solution onto the base plate by using the pin.Therefore, the shape of the spot is diversified, for example, due to theshape of the forward end of the pin and/or the residue of the samplesolution remaining at the forward end of the pin after the supply. Asshown in FIG. 18, spots 200, each of which has many irregularities atthe outer circumferential portion, are formed on a base plate 202.

When unknown DNA is inspected by using a DNA microarray arranged with alarge number of spots having dispersed shapes, it is apt to be difficultto recognize the fluorescence light emission from the spot with a CCDcamera or the like. Therefore, the inspection accuracy may be lowered.

Further, when many irregularities exist at the outer circumferentialportion, the sample solution flows through angular portions. Therefore,the sample solutions in the plurality of spots 200 may be mixed witheach other.

The present invention has been made taking the foregoing problems intoconsideration, an object of which is to provide a DNA microarray whichmakes it possible to improve the inspection accuracy for geneticanalyses and which makes it possible to increase the amount ofinformation to be obtained.

Another object of the present invention is to provide a DNA microarraywhich makes it possible to achieve a high degree of concentration ofspots and which makes it possible to perform detailed genetic analyses.

Still another object of the present invention is to provide a DNAmicroarray which makes it possible to recognize the degree of thereaction with respect to an amount of DNA fragments immobilized in aspot and which makes it possible to obtain an analog inspection resultfor a specimen, in addition to a digital inspection result to indicatewhether or not the reaction occurs.

The applicable range of the present invention is not limited to the DNAmicroarray in which DNA fragments are aligned and immobilized as spots.The present invention is generally usable for every type of the biochipwhich specifically reacts with a biochemical specimen and which is usedin order to obtain information on the structure of the specimen.

SUMMARY OF THE INVENTION

The present invention lies in a biochip comprising a large number ofspots based on capture solutions arranged on a base plate, obtained bysupplying, onto the base plate, a plurality of types of the capturesolutions each of which specifically reacts with a specimen and each ofwhich is used to obtain information on a structure of the specimen;wherein a plurality of the spots, which have different spot sizes, areformed on the base plate.

Accordingly, it is possible for the respective spots to suppress thedispersion of the ability to capture the specimen among the spots, whichwould be otherwise caused by the difference in amount of the captureimmobilized on the spot or by the different abilities of the captures tocapture the specimen. Thus, it is possible to suppress the dispersion ofinspection results and the deterioration of quantitative performance,which would be otherwise caused by the difference in detectionsensitivity among the spots.

That is, the spot, which corresponds to the capture with a small amountto be immobilized on the base plate or which corresponds to the capturewith a low ability to capture the specimen, is increased in size,generally in diameter of a circular configuration. Accordingly, thedetection sensitivity per one spot can be increased. As a result, it ispossible to uniformize the detection sensitivities of all of the spots.

In another aspect, the present invention has the following feature. Thatis, when a plurality of the spots are formed for captures of anidentical type on a single sheet of the base plate, then the pluralityof the spots, which have different spot sizes on the base platerespectively, are formed for the captures of the identical type.

When the construction as described above is adopted, it is possible torecognize the degree of the reaction corresponding to the size of thespot, in addition to a digital inspection result to indicate whether ornot the reaction occurs with respect to the captures of the identicaltype. Thus, it is possible to obtain an analog inspection result for thespecimen. Of course, the analog inspection result can be theoreticallyobtained by detecting, in an analog manner, the amount of a probe whichreacts with the capture immobilized in one spot. However, actually, sucha procedure cannot be executed due to the restriction including, forexample, the detection sensitivity of the detection equipment, theresolution, and the reaction efficiency. Therefore, the analog analysiscan be performed by combining the plurality of spots using the pluralityof spots having the different sizes of the spots on the base platerespectively for the captures of the identical type as performed in thepresent invention, although the detection of a each spot is performed ina digital manner.

In still another aspect, the present invention lies in a biochipcomprising a large number of spots based on capture solutions arrangedon a base plate, obtained by supplying, onto the base plate, a pluralityof types of the capture solutions each of which specifically reacts witha specimen and each of which is used to obtain information on astructure of the specimen; wherein a plurality of the spots are formed,in which an amount of a capture per unit area immobilized in each of thespots differs.

Accordingly, it is possible for the respective spots to suppress thedispersion of the ability to capture the specimen among the spots, whichwould be otherwise caused by the different abilities of the captures tocapture the specimen, in the same manner as in the case in which thesizes of the spots differ as described above. Thus, it is possible tosuppress the dispersion of inspection results and the deterioration ofquantitative performance, which would be otherwise caused by thedifference in detection sensitivity among the spots. That is, theconcentration of the capture solution to be supplied is increased forthe spot which corresponds to the capture with a low ability to capturethe specimen. Accordingly, the amount of the capture immobilized on thespot is increased per unit area, and the detection sensitivity per onespot is increased. As a result, it is possible to uniformize thedetection sensitivities of all of the spots.

The method of changing the amount per unit area of the capture amountimmobilized on one spot may be also carried out by changing theconcentration of the capture solution to be supplied as described above.Alternatively, the method may be also carried out by changing thecapture amount to be supplied to one spot.

There is a certain upper limit for the capture amount immobilized perone spot. Therefore, the capture solution having a concentration lowerthan an average of all spots, or the capture solution in an amountsmaller than an average of all spots is supplied for the spotcorresponding to the capture having the high ability to capture thespecimen. On the other hand, the capture solution at a concentrationand/or in an amount corresponding to the upper limit of the captureamount to be immobilized or corresponding to an amount exceeding theupper limit is supplied to the spot corresponding to the capture havingthe low ability to capture the specimen.

Mistakes tend to be caused when the concentration and the amount of thecapture solution to be supplied are individually managed for therespective spots as described above. It is advantageous to simplify thestep to be as simple as possible. In such a case, when the capturesolution is supplied onto the base plate by using an ink-jet method asdescribed later on, it is preferable that the amount of solution to besupplied is changed by changing the number of discharge times for onespot.

The method of suppressing the dispersion of the ability to capture thespecimen among the spots caused by the captures having the differentabilities to capture the specimen by changing the concentration of thecapture solution to be supplied or by changing the amount of the captureto be supplied for one spot is also used to reduce the dispersion whenthe immobilization ratio of the capture to be immobilized per one spotdiffers.

That is, as for the formation of the spot corresponding to the capturehaving the low immobilization ratio, it is possible to suppress thedispersion of the immobilization efficiency among the respective spotsby increasing the concentration of the capture solution to be supplied,or by increasing the amount of the capture solution to be supplied perone spot.

In still another aspect of the present invention, when a plurality ofspots of the captures of an identical type are formed on one sheet ofthe base plate, the plurality of the spots, which have different amountsof the capture per unit area immobilized on the base plate respectively,are formed for the captures of the identical type.

When the construction as described above is adopted, it is possible torecognize the degree of the reaction corresponding to the amount of thecapture immobilized per unit area of the spot, in addition to a digitalinspection result to indicate whether or not the reaction occurs withrespect to the capture, concerning the captures of the identical type inthe same manner as in the case in which the sizes of the spots differ asdescribed above. Thus, it is possible to obtain an analog inspectionresult for the specimen. Of course, the analog inspection result can betheoretically obtained by detecting, in an analog manner, the amount ofa probe which reacts with the capture immobilized in one spot. However,actually, such a procedure cannot be executed due to the restrictionincluding, for example, the detection sensitivity of the detectionequipment, the resolution, and the reaction efficiency. Therefore, theanalog analysis can be performed by combining the plurality of spots,although the detection itself is performed in a digital manner with theplurality of spots having the different amounts of the captureimmobilized per unit area of each of the spots on the base platerespectively for the captures of the identical type as performed in thepresent invention.

In still another aspect, the present invention lies in a biochipcomprising a large number of spots based on capture solutions arrangedon a base plate, obtained by supplying, onto the base plate, a pluralityof types of the capture solutions each of which specifically reacts witha specimen and each of which is used to obtain information on astructure of the specimen; wherein the spots, which are composed ofdifferent types of the captures, are formed at an identical spotformation position. In this case, it is possible to greatly reduce thearrangement area for the spots, and it is possible to miniaturize thebiochip itself.

In still another aspect, the present invention lies in a biochipcomprising a large number of spots based on capture solutions arrangedon a base plate, obtained by supplying, onto the base plate, a pluralityof types of the capture solutions each of which specifically reacts witha specimen and each of which is used to obtain information on astructure of the specimen; wherein each of the spots has a shape of asubstantially circular configuration, and a ratio between a major axisand a minor axis of the substantially circular configuration is not lessthan 0.9 and not more than 1.1.

Accordingly, the dispersion of the shape of each of the spots isreduced. It is easy to recognize the fluorescence light emission fromthe spot with a CCD camera or the like, and the inspection accuracy isimproved. Especially, owing to the fact that the planar configuration ofthe spot is substantially circular, it is possible to avoid flowing thesample solution from the spot during the formation of the spot, and itis possible to prevent the sample solutions in the plurality of spotsfrom being mixed with each other. In this case, it is also preferablethat the spots are arranged at least in a zigzag configuration, and aratio of an area in which the spot is not deposited with respect to aninspection effective area on the base plate is not more than 22%. Inthis case, it is possible to achieve a high degree of concentration ofspots. Accordingly, it is possible to perform detailed genetic analysisfor a large amount of a biochemical sample at once.

It is preferable for the biochip described above that the spots based onthe sample solution are formed by means of an ink-jet system.

In the ink-jet system, the spot is formed by discharging the capturesolution into the atmospheric air and allowing the capture solution toarrive at the base plate as a target. Therefore, the shape of the spotis a circular configuration which is approximate to a perfect circleowing to the surface tension of the sample. Therefore, the dispersion ofthe shape is reduced for the respective spots. Owing to the fact thatthe force of discharge and the number of times of discharge per unittime (discharge frequency) can be electrically controlled, the amount ofthe capture supplied to one spot on the base plate can be freelychanged. Thus, the size of the spot and the amount of the capture perunit volume immobilized in the spot on the base plate can be varied.

Especially, the amount of the capture per unit volume is preferablyvaried by discharging and supplying the capture solution a plurality oftimes to one spot on the base plate in accordance with the ink-jetsystem. That is, the capture solution is discharged and supplied aplurality of times in a divided manner without discharging and supplyinga large amount of the capture solution at once. Further, the dischargeinterval is adjusted so that a previously formed spot is not widened inspot diameter due to superimposition of the capture solutionsubsequently discharged. Accordingly, the amount of the capture suppliedto the spot can be increased or decreased without changing the size ofthe spot. Thus, it is possible to vary the capture density per unitarea.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view illustrating a DNA microarray accordingto an embodiment of the present invention;

FIG. 2 shows a magnified sectional view illustrating an arrangement ofthe DNA microarray according to the embodiment of the present invention;

FIG. 3 shows a block diagram illustrating steps of a method of producingthe DNA microarray according to the embodiment of the present invention;

FIG. 4 shows a block diagram illustrating steps as contents of a samplepreparation step;

FIG. 5A shows a plan view illustrating an arrangement of a dispenser tobe used for the method of producing the DNA microarray according to afirst embodiment;

FIG. 5B shows a front view thereof;

FIG. 5C shows a magnified plan view illustrating one micropipette forconstructing the dispenser;

FIG. 6 shows a longitudinal sectional view illustrating an arrangementof the micropipette;

FIG. 7 shows the shape of a flow passage including a cavity formed in asubstrate of the micropipette;

FIG. 8 shows an exploded perspective view illustrating the dispensertogether with a cartridge;

FIG. 9 illustrates a first method adopted when the DNA microarray isproduced by using the dispenser;

FIG. 10 illustrates a second method adopted when the DNA microarray isproduced by using the dispenser;

FIG. 11 illustrates a state of formation of a spot of the DNA microarrayaccording to the embodiment of the present invention;

FIG. 12 illustrates a state in which spots are arranged in a matrixform;

FIG. 13 illustrates a state in which spots are arranged in a zigzagform;

FIG. 14A illustrates a state in which a plurality of spots havingdifferent spot sizes are formed on a base plate;

FIG. 14B illustrates a state in which four spots having different sizesrespectively are formed for an identical DNA fragment;

FIG. 15A illustrates a state in which a plurality of spots are formed,in which the amount of the capture per unit area immobilized in each ofthe spots differs:

FIG. 15B illustrates a state in which four spots are formed for anidentical DNA fragment, in which the amount of the immobilized captureper unit area differs respectively;

FIG. 16 illustrates a state in which different types of spots are formedat an identical spot formation position;

FIG. 17 shows a sectional view taken along a line XVII—XVII shown inFIG. 16; and

FIG. 18 illustrates shapes of conventional spots.

DETAILED DESCRIPTION OF THE DRAWINGS

Embodiments of the DNA microarray according to the present inventionwill be explained below with reference to FIGS. 1 to 18.

As shown in FIGS. 1 and 2, a DNA microarray 20 according to anembodiment of the present invention comprises a large number of minutespots 80 arranged on a base plate 10 by supplying (including dropwiseaddition) a sample solution. A poly-L-lysine layer 12 is formed on thesurface of the base plate 10.

The DNA microarray 20 is produced by forming the minute spots 80 bysupplying the sample solution onto the base plate 10, for example, byperforming production steps as shown in FIG. 3.

That is, the DNA microarray 20 is produced by performing a pretreatmentstep S1 of forming the poly-L-lysine layer 12 (see FIG. 2) on thesurface of the base plate 10, a sample preparation step S2 of preparingthe sample solution containing DNA fragment, and a supply step S3 ofsupplying the obtained sample solution onto the base plate 10.

As shown in FIG. 4, the sample preparation step S2 includes anamplification step S11 of performing PCR amplification for the DNAfragment to prepare a PCR product, a purification step S12 of purifyingand drying the obtained PCR product to prepare DNA powder, and a mixingstep S13 of dissolving the obtained DNA powder in a buffer solution.

The process will be specifically explained below. That is, in thepretreatment step S1, the base plate 10 is firstly immersed in analkaline solution to perform slow shaking at room temperature for atleast 2 hours. The alkaline solution is a solution which is obtained,for example, by dissolving NaOH in distilled water, and adding ethanolthereto, followed by being agitated until the solution is completelytransparent.

After that, the base plate 10 is taken out, and it is transferred intodistilled water, followed by being rinsed to remove the alkalinesolution. Subsequently, the base plate 10 is immersed in a poly-L-lysinesolution prepared by adding poly-L-lysine to distilled water, followedby being left to stand for 1 hour.

After that, the base plate 10 is taken out, and it is applied to acentrifugal machine to perform centrifugation so that any excessivepoly-L-lysine solution is removed. Subsequently, the base plate 10 isdried at 40° C. for about minutes to obtain the base plate 10 with thepoly-L-lysine layer 12 formed on the surface.

Subsequently, the sample preparation step S2 is performed. At first, 3Msodium acetate and isopropanol are added to the PCR product amplified byusing a known PCR machine (amplification step S11), followed by beingleft to stand for several hours. After that, the PCR product solution iscentrifuged with a centrifugal machine to precipitate the DNA fragments.

The precipitated DNA fragments are rinsed with ethanol, followed bycentrifugation. After that, the DNA fragments are dried to produce theDNA powder (purification step S12). A certain amount of ×1 TE buffer isadded to the obtained DNA powder, followed by being left to stand forseveral hours to completely dissolve the DNA powder (mixing step S13).Thus, the sample solution is prepared. The concentration of the samplesolution at this stage is 0.1 to 10 μg/ml.

In the embodiment of the present invention, the obtained sample solutionis supplied onto the base plate 10 to produce the DNA microarray 20(supply step S3). An immobilizing solution may be mixed with the samplesolution obtained by performing the sample preparation step S2. Thesample solution may be diluted as well. In this case, the buffersolution described above, an aqueous solution containing water and NaCl,or an aqueous solution containing polymer may be used as a dilutingsolution.

When the DNA microarray 20 is produced in this embodiment, for example,a disperser 30 shown in FIGS. 5A to 7 is effectively used.

As shown in FIGS. 5A and 5B, the dispenser 30 has the followingarrangement. That is, for example, ten micropipettes 34 are arranged infive rows and two columns on an upper surface of a fixation plate 32having a rectangular configuration. A group of the micropipettes 34arranged in the direction of each column are fixed on the fixation plate32 by the aid of a fixing jig 36 respectively.

As shown in FIGS. 5C and 6, the micropipette 34 comprises asample-pouring port 52 which is formed at the upper surface of asubstrate 50 having a substantially rectangular parallelepiped-shapedconfiguration, a sample discharge port 54 which is formed at the lowersurface of the substrate 50, a cavity 56 which is formed between thesample-pouring port 52 and the sample discharge port 54, and an actuatorsection 58 which is used to vibrate the substrate 50 or change thevolume of the cavity 56.

Therefore, as shown in FIG. 6, through-holes 40 are provided through thefixation plate 32 at portions corresponding to the sample dischargeports 54 of the micropipettes 34 respectively. Accordingly, the samplesolution, which is discharged from the sample discharge port 54 of themicropipette 34, is supplied through the through-hole 40, for example,to the base plate 20 which is fixed under the fixation plate 32.

An introducing bore 60 having a substantially L-shaped configurationwith a wide opening is formed over a region ranging from thesample-pouring port 52 to the inside of the substrate 50 in themicropipette 34. A first communication hole 62 having a small diameteris formed between the introducing bore 60 and the cavity 56. The samplesolution, which is poured from the sample-pouring port 52, is introducedinto the cavity 56 through the introducing bore 60 and the firstcommunication hole 62.

A second communication hole 64, which communicates with the sampledischarge port 54 and which has a diameter larger than that of the firstcommunication hole 62, is formed at a position different from that ofthe first communication hole 62, of the cavity 56. In the embodiment ofthe present invention, the first communication hole 62 is formed at theportion of the lower surface of the cavity 56 deviated toward thesample-pouring port 52. The second communication hole 64 is formed atthe position of the lower surface of the cavity 56 as well correspondingto the sample discharge port 54.

Further, in the this embodiment, the portion of the substrate 50, withwhich the upper surface of the cavity 56 makes contact, is thin-walledto give a structure which tends to undergo the vibration with respect tothe external stress so that the portion functions as a vibrating section66. The actuator section 58 is formed on the upper surface of thevibrating section 66.

The substrate 50 is constructed by laminating a 5 plurality of greensheets made of zirconia ceramics (first thin plate layer 50A, firstspacer layer 50B, second thin plate layer 50C, second spacer layer 50D,and third thin plate layer 50E) followed by being sintered into oneunit.

That is, the substrate 50 is constructed by laminating the thin-walledfirst thin plate layer 50A which is formed with a window forconstructing the sample-pouring port 52 and which constitutes a part ofthe vibrating section 66, the thick-walled first spacer layer 50B whichis formed with a part of the introducing bore 60 and a plurality ofwindows for constructing the cavity 56 respectively, the thin-walledsecond thin plate layer 50C which is formed with a part of theintroducing bore 60 and a plurality of windows for constructing parts ofthe second communication hole 64 and the first communication hole 62respectively, the thick-walled second spacer layer 50D which is formedwith a plurality of windows for constructing a part of the introducingbore 60 and a part of the second communication hole 64 respectively, andthe thin-walled third thin plate layer 50E which is formed with a windowfor constructing the sample discharge port 54, followed by beingsintered into one unit.

The actuator section 58 is constructed to have the vibrating section 66described above as well as a lower electrode 70 which is directly formedon the vibrating section 66, a piezoelectric layer 72 which is composedof, for example, a piezoelectric/electrostrictive layer or ananti-ferroelectric layer formed on the lower electrode 70, and an upperelectrode 74 which is formed on the upper surface of the piezoelectriclayer 72.

As shown in FIG. 5C, the lower electrode 70 and the upper electrode 74are electrically connected to an unillustrated driving circuit via aplurality of pads 76, 78 which are formed on the upper surface of thesubstrate 50 respectively.

The micropipette 34 constructed as described above is operated asfollows. That is, when an electric field is generated between the upperelectrode 74 and the lower electrode 70, then the piezoelectric layer 72is deformed, and the vibrating section 66 is deformed in accordancetherewith. Accordingly, the volume of the cavity (pressurizing chamber)56 contacting with the vibrating section 66 is decreased.

When the volume of the cavity 56 is decreased, the sample solutioncharged in the cavity 56 is discharged at a predetermined speed from thesample discharge port 54 which communicates with the cavity 56. As shownin FIG. 1, it is possible to prepare the DNA microarray 20 in which thesample solutions discharged from the micropipettes 34 are aligned andfixed as minute spots 80 on the base plate 50 such as a microscopicslide glass.

In this arrangement, when the arrangement pitch of the sample dischargeports 54 in the dispenser 30 is larger than the arrangement pitch of theminute spots 80 formed on the base plate 10, the sample solution issupplied while shifting the supply position for the dispenser 30.

An apparatus structure based on the so-called ink-jet system may beadopted as the structure in which the volume of the cavity 56 isdecreased in accordance with the driving of the actuator section 58 (seeJapanese Laid-Open Patent Publication No. 6-40030).

The cavity (pressurizing chamber) 56 is preferably formed to have such aflow passage dimension that the sample solution containing DNA fragmentsor the like is moved in laminar flow.

That is, the dimension of the cavity 56 differs depending on the type ofthe sample, the size of liquid droplets to be prepared, and the densityof formation. However, for example, when DNA fragments of base pairshaving a length of about 1 to 10,000 bp are dissolved in a buffersolution (TE buffer) at a concentration of 0.5 μg/μliter to obtain asample which is dripped at a pitch of several hundreds μm to give aliquid droplet diameter of several hundreds μmφ, then it is preferablethat the cavity length (L) is 1 to 5 mm, the cavity width (W) is 0.1 to1 mm, and the cavity depth (D) is 0.1 to 0.5 mm as shown in FIG. 7. Itis preferable that the inner wall of the cavity 56 is smooth withoutinvolving any projection to disturb the flow. It is preferable that thematerial of the cavity 56 is made of ceramics which has good affinitywith respect to the sample solution.

When the shape as described above is adopted, the cavity 56 can be usedas a part of the flow passage ranging from the sample-pouring port 52 tothe sample discharge port 54. The sample can be introduced to the sampledischarge port 54 without disturbing the flow of the sample solutionwhich is moved from the sample-pouring port 52 via the introducing bore60 and the first communication hole 62 to the inside of the cavity 56.

As shown in FIG. 5A, a plurality of pins 38 for positioning and fixingthe micropipettes 34 are provided on the upper surface of the fixationplate 32. When the micropipette 34 is fixed on the fixation plate 32,the micropipette 34 is placed on the fixation plate 32 while insertingthe pins 38 of the fixation plate 32 into positioning holes 90 (see FIG.5C) provided at the both sides of the substrate 50 of the micropipette34. Thus, a plurality of micropipettes 34 are automatically positionedwith a predetermined array arrangement.

Each of the fixing jigs 36 has a holder plate 100 for pressing theplurality of micropipettes 34 against the fixation plate 32. Insertionholes for inserting screws 102 thereinto are formed through both endportions of the holder plate 100. When the screws 102 are inserted intothe insertion holes, and they are screwed into the fixation plate 32,then the plurality of micropipettes 34 can be pressed against thefixation plate 32 by the aid of the holder plate 100 at once. One unitis constructed by the plurality of micropipettes 34 which are pressed byone holder plate 100. The example shown in FIG. 5A is illustrative ofthe case in which one unit is constructed by the five micropipettes 34which are arranged in the direction of the column.

The holder plate 100 is formed with introducing holes 104 (see FIG. 5B)which are used to supply the sample solutions to the portionscorresponding to the sample-pouring ports 52 of the respectivemicropipettes 34 respectively when the plurality of micropipettes 34 arepressed. Tubes 106 for introducing the sample solution to theintroducing holes 104 respectively are held at upper end portions of therespective introducing holes 104.

Considering the realization of the efficient wiring operation, it ispreferable that the width of the holder plate 100 resides in such adimension that the pads 76, 78 connected to the respective electrodes70, 74 of the actuator section 58 are faced upwardly when the pluralityof micropipettes 34 are pressed against the fixation plate 32.

As described above, the dispenser 30 described above is constructed suchthat the plurality of micropipettes 34 each having the sample-pouringport 52 and the sample discharge port 54 are provided in an upstandingmanner with the respective sample discharge ports 54 directeddownwardly.

That is, the respective micropipettes 34 are aligned and arranged suchthat the respective sample-pouring ports 52 are disposed on the upperside, the sample discharge ports 54 are disposed on the lower side, andthe respective sample discharge ports 54 are aligned two-dimensionally.Sample solutions of mutually different types are discharged from thesample discharge ports 54 respectively.

When the dispenser 30 constructed as described above is used, anautomatic dispenser or the like, which is constructed by combining an XYrobot and the dispenser, is generally used for a method of supplying thesample solutions of mutually different types corresponding to therespective sample-pouring ports 52. However, as shown in FIG. 8, forexample, a method is available, which is based on the use of a cartridge112 arranged with a large number of recesses (storage sections) 110 eachhaving a substantially V-shaped cross section. For this method, forexample, the following procedure is available. That is, the samplesolutions of the different types are poured into the respective recesses110 of the cartridge 112 respectively. The cartridge 112 is attached sothat the respective recesses 110 correspond to the tubes 106respectively. The bottoms of the respective recesses 110 are opened withneedles or the like. Accordingly, the sample solutions in the respectiverecesses 110 are supplied via the tubes 106 to the respectivemicropipettes 34.

When the tubes 106 are not used, for example, the following method isavailable. That is, the cartridge 112 is attached so that the respectiverecesses 110 correspond to the respective introducing holes 104 of thefixing jig 36 respectively. The bottoms of the respective recesses 110are opened with needles or the like. Accordingly, the sample solutionsin the respective recesses 110 are supplied via the introducing holes104 to the respective micropipettes 34. Alternatively, needles or thelike may be formed in the vicinity of the respective introducing holes104 of the fixing jig 36 beforehand so that the respective recesses 110may be opened simultaneously with the attachment of the cartridge 112 tothe fixing jig 36.

Alternatively, it is also preferable to add a mechanism for feeding thegas or the like under the pressure after the opening to forcibly extrudethe sample solutions. Further alternatively, it is also preferable toadd a mechanism for making aspiration from the discharge ports of therespective micropipettes. It is desirable to provide a mechanism forwashing the space ranging from the sample-pouring port 52 to the sampledischarge port 54 formed in the substrate 50 of each of themicropipettes 34, for example, in order that several thousands toseveral tens thousands types or many kinds of DNA fragments aredischarged as the minute spots 80 with good purity without involving anycontamination.

In the example shown in FIG. 5A, the both ends of the holder plate 100are tightened to the fixation plate 20 by the aid of the screws 102.However, the holder plate 100 may be fixed in accordance with othermethods based on the mechanical procedure by using, for example, anadhesive or the like, as well as screws and springs.

As described above, the substrate 50 for constructing the micropipette34 is formed of ceramics, for which it is possible to use, for example,fully stabilized zirconia, partially stabilized zirconia, alumina,magnesia, and silicon nitride.

Among them, the fully stabilized/partially stabilized zirconia is usedmost preferably, because the mechanical strength is large even in thecase of the thin plate, the toughness is high, and the reactivity withthe piezoelectric layer 72 and the electrode material is low.

When the fully stabilized/partially stabilized zirconia is used as thematerial, for example, for the substrate 50, it is preferable that atleast the portion (vibrating section 66), on which the actuator section58 is formed, contains an additive such as alumina and titania.

Those usable as the piezoelectric ceramic for the piezoelectric layer 72for constructing the actuator section 58 include, for example, leadzirconate, lead titanate, lead magnesium niobate. lead magnesiumtantalate, lead nickel niobate, lead zinc niobate, lead manganeseniobate, lead antimony stannate, lead manganese tungstate, lead cobaltniobate, and barium titanate, as well as composite ceramics containingcomponents obtained by combining any of them. However, in the embodimentof the present invention, a material containing a major componentcomposed of lead zirconate, lead titanate, and lead magnesium niobate ispreferably used, for following reason.

That is, such a material has a high electromechanical coupling factorand a high piezoelectric constant. Additionally, such a material has lowreactivity with the substrate material during the sintering of thepiezoelectric layer 72, making it possible to stably form the producthaving a predetermined composition.

Further, in the embodiment of the present invention, it is alsopreferable to use ceramics obtained by appropriately adding, to thepiezoelectric ceramics described above, for example, oxides oflanthanum, calcium, strontium, molybdenum, tungsten, barium, niobium,zinc, nickel, manganese, cerium, cadmium, chromium, cobalt, antimony,iron, yttrium, tantalum, lithium, bismuth, and stannum, or a combinationof any of them, or other compounds.

For example, it is also preferable to use ceramics containing a majorcomponent composed of lead zirconate, lead titanate, and lead magnesiumniobate, and further containing lanthanum and/or strontium.

On the other hand, it is preferable that the upper electrode 74 and thelower electrode 70 of the actuator section 58 are made of metal which issolid at room temperature and which is conductive. For example, it ispossible to use metal simple substance of, for example, aluminum,titanium, chromium, iron, cobalt, nickel, copper, zinc, niobium,molybdenum, ruthenium, palladium, rhodium, silver, stannum, tantalum,tungsten, iridium, platinum, gold, and lead, or alloy obtained bycombining any of them. It is also preferable to use a cermet materialobtained by dispersing, in the metal described above, the same materialas that of the piezoelectric layer 72 or the substrate 50.

Next, explanation will be made with reference to FIGS. 9 to 12 forseveral methods for producing the DNA microarray 20 by using thedispenser 30.

At first, a first method is shown in FIG. 9. That is, mutually differenttypes of sample solutions are charged from the respective tubes 106 viathe introducing holes 104 of the fixing jig 36 into the cavities 56 ofthe respective micropipettes 34 respectively. Subsequently, therespective actuator sections 58 are driven to discharge the samplesolutions from the sample discharge ports 54 of the respectivemicropipettes 34. As for the method of charging the solution into thecavity 56, the solution may be poured in accordance with the capillaryforce of the solution introduced from the sample-pouring port 52.However, it is reliable to adopt a method in which the solution ischarged by means of vacuum aspiration through the sample discharge port54.

As for the voltage waveform to be applied to the respective electrodes70, 74 of the actuator section 58, when the actuator section 58 issubjected to the ON operation to decrease the volume of the cavity 56, apulsed voltage is applied to the respective electrodes 70, 74. In thiscase, the deformation of the vibrating section 66 is increased byincreasing the amplitude of the pulse, and the discharge force and thedischarge amount of the sample solution are also increased correspondingthereto. When a plurality of pulses are applied for a certain period, alarge number of sample solutions each having a small amount can bedischarged by shortening the pulse cycle and decreasing the amplitude ofeach pulse.

Especially, when it is required to improve the accuracy of the controlof the amount of the sample to be supplied per one spot when the DNAmicroarray is produced by using a plurality of micropipettes, it ispreferable to adopt the method of discharging a large number of samplesolutions in a small amount, for the following reason. That is, thenumber of times of discharge can be completely controlled electrically,and hence the minute dispersion of the discharge ability (dischargeamount) for each of the micropipettes can be corrected by the number oftimes of discharge.

During this process, when the supply position is appropriately changed,the droplets of the supplied sample solution are combined (integrated)on the base plate 10 to form the sample solution having one spotdiameter. Further, it is possible to realize a uniform spot diameterformed on the base plate 10 by controlling the number of supplyoperations, the supply position, and the amount of one time supply,depending on the type of the sample solution to be supplied.

Next, explanation will be made for a second method based on the use ofthe dispenser 30. The second method is shown in FIG. 10. That is, asubstitution solution such as a buffer solution, an aqueous solutioncontaining NaCl, and an aqueous solution containing polymer is chargedinto the cavity 56 of each of the micropipettes 34 from each of thetubes 106 via the introducing hole 104 of the fixing jig 36respectively. Subsequently, the sample is poured into the cavity 56 fromthe sample-pouring port 52 while effecting the laminar flow substitutionto wait for the completion of the substitution thereafter. After that,the actuator section 58 is driven to discharge and supply the samplesolution onto the base plate 10.

It is preferable that the completion of the laminar flow substitution ofthe sample in the cavity 56 is recognized by sensing the change of thefluid characteristic in the cavity 56.

It is preferable that the substitution between the substitution solutionand the sample solution in the cavity 56 is performed in a form of thelaminar flow. However, when the type of the sample is changed, or whenthe movement speed of the liquid is extremely fast, then it is notnecessarily indispensable to use the laminar flow at portions of thecavity 56 in the vicinity of the first communication hole 62. In thiscase, the purge amount of the sample solution is increased due to themixing of the sample and the substitution solution. However, it ispossible to suppress the increase in the purge amount to be minimum byjudging the completion of the substitution by sensing the change of thefluid characteristic in the cavity 56.

In the present invention, the change of the fluid characteristic in thecavity 56 is recognized by applying a voltage in such a degree as toexcite the vibration in the actuator section 58, and detecting thechange of the electric constant caused by the vibration. Such aprocedure for sensing the change of the fluid characteristic isdisclosed, for example, in Japanese Laid-Open Patent Publication No.8-201265.

Specifically, the electric connection from a power source for drivingthe discharge is separated from the actuator section 58 at apredetermined interval by using a relay. Simultaneously, a means formeasuring the resonance frequency is connected by using the relay. Atthis point of time, the impedance or the resonance characteristic suchas the resonance frequency or the attenuation factor is electricallymeasured.

Accordingly, it is possible to recognize, for example, whether or notthe viscosity and the specific gravity of the liquid are those of theobjective sample (liquid containing the DNA fragment or the like). Thatis, as for each of the micropipettes 34, the micropipette 34 itselffunctions as a sensor. Therefore, it is also possible to simplify thestructure of the micropipette 34.

The actuator section 58 is driven under a driving conditioncorresponding to the amount of liquid droplets suitable for the requiredspot diameter, and the sample solution is repeatedly supplied.Accordingly, the DNA microarray 20 is produced. Usually, when one minutespot 80 is formed, one to several hundreds of droplet or droplets aredischarged from the micropipette 34.

When the amount of the sample in the sample-pouring port 52 isdecreased, the discharge is continued by adding the buffer solution sothat no bubble enters the inside of the flow passage. Accordingly, allof the sample solution can be used without allowing the sample solutionto remain in the micropipette 34. The completion of the substitutionfrom the sample to the substitution solution (completion of the sampledischarge) is confirmed by detecting the viscosity and the specificgravity of the liquid by using the actuator section 58 in the samemanner as described above.

It is preferable to use the substitution solution and the samplesolution such that the dissolved gas in the solution is previouslyremoved by performing the degassing operation. When such a solution isused, if any bubble obstructs the flow passage at an intermediateportion to cause the defective charge upon the charge of the solutioninto the flow passage of the micropipette 34, then the inconvenience canbe avoided by dissolving the bubble in the solution. Further, no bubbleis generated in the fluid during the discharge, and no defectivedischarge is caused as well.

In the second method described above, the substitution solution such asa buffer solution, an aqueous solution containing NaCl, and an aqueoussolution containing polymer is poured from the sample-pouring port 52into the cavity 56 while discharging the sample solution, and the samplesolution remaining in the cavity 56 is completely discharged for pouringthe next sample.

When it is sensed whether or not the sample solution remains in thecavity 56 (whether or not the discharge can be effected as the samplesolution), the recognition can be also made by sensing the change of thefluid characteristic in the cavity 56. In this case, a mechanism fordetecting the completion of the substitution can be used to extremelydecrease the purge amount of the sample which is not used and improvethe efficiency of the use of the sample solution.

It is also preferable that when the sample is charged from thesample-pouring port 52 to the cavity 56, the interior of the cavity 56is substituted with the sample from the sample-pouring port 52 whiledriving the actuator section 58. In this procedure, the interior of thecavity 56 can be substituted in a reliable manner with the inexpensivesubstitution solution beforehand. As a result, it is possible tocompletely avoid the occurrence of any defective discharge, and it ispossible to efficiently discharge the expensive sample.

Further, the following procedure may be adopted. That is, thesubstitution solution such as a buffer solution, an aqueous solutioncontaining NaCl, and an aqueous solution containing polymer is chargedinto the cavity 56. The amount of the substitution solution existing inthe cavity 56 and in the sample-pouring port 52 is adjusted to be apredetermined amount. Subsequently, a predetermined liquid amount of thesample solution is poured from the sample-pouring port 52, and then theactuator section 58 is driven in an amount corresponding to apredetermined number of pulses to discharge the amount of thesubstitution solution existing in the cavity 56 and in thesample-pouring port 52.

In this manner, the amount of the substitution solution existing in thecavity 56 and in the sample-pouring port 52 is correctly discharged, andit is possible to complete the charge of the sample solution without anyloss.

In the first and second methods described above, for example, as shownin FIG. 11, the planar configuration of the spot 80 formed on the baseplate 10 is substantially circular. In this case, the ratio between themajor axis La and the minor axis Lb of each of the spots 80 is not lessthan 0.9 and not more than 1.1.

Accordingly, the dispersion of the shapes of the respective spots 80 isreduced. Even when unknown DNA is inspected, it is easy to recognize thefluorescence light emission from the spot 80 with a CCD camera or thelike. Thus, the inspection accuracy is improved. Especially, since theplanar configuration of the spot 80 is substantially circular, it ispossible the avoid the flow of the sample solution during the formationof the spot 80, and it is possible to prevent the sample solutions inthe plurality of spots 80 from being mixed with each other.

In the embodiment of the present invention, as shown in FIG. 12, when alarge number of spots 80 are arranged, the spots 80 can be concentratedup to positions at which the adjoining spots 80 make contact with eachother. Further, as shown in FIG. 13, a large number of spots 80 can bealso arranged in a zigzag configuration. In this case, when the spots 80are concentrated up to positions at which the adjoining spots 80 makecontact with each other, the ratio of the non-deposition area Ab of thespots 80 (area Ab of the portion at which the spots 80 are not formed)with respect to the inspection effective area Aa (area Aa of thesubstantially rectangular region in which the spots 80 are arranged) onthe base plate 10 is not more than about 9%.

As described above, in the embodiment of the present invention, it ispossible to achieve the high concentration of the spots 80. Therefore,it is possible to perform the detailed genetic analysis for a largeamount of the sample at once.

In the embodiment of the present invention, as shown in FIG. 14A, aplurality of spots having different spot sizes can be formed on the baseplate. In the example shown in FIG. 14A, when different amounts of DNAare immobilized on the spots, or when different species of DNA havingdifferent efficiencies of hybridization with a specimen are immobilizedfor the respective spots 1A to 3D with different types of DNA fragments,then the size of the spot, i.e., the diameter of the circularconfiguration in general is changed.

Specifically, the spots are formed as follows. That is, the spotdiameter is large for each of the spots 1A, 1D, 2B, 2C, 2D, 3A, 3D inwhich the amount of DNA immobilized on the spot is small, or in whichthe DNA species having a low efficiency of hybridization with thespecimen is immobilized. The spot diameter is intermediate for each ofthe intermediate spots 1B, 2A, 3B. The spot diameter is small for eachof the spots 1C, 3C in which the amount of DNA immobilized on the spotis large, or in which the DNA species having a high efficiency ofhybridization with the specimen is immobilized. Therefore, it ispossible to suppress the dispersion of the ability to capture thespecimen. Further, it is possible to suppress the deterioration of thequantitative performance and the dispersion of the inspection resultwhich would be otherwise caused by the difference in detectionsensitivity between the spots.

In the embodiment of the present invention, as shown in FIG. 14B, aplurality of spots having different sizes (spot diameters) respectivelycan be formed for an identical. DNA fragment. The example shown in FIG.14B is illustrative of a state in which four spots A₁ to A₄ havingdifferent sizes (spot diameters) respectively are formed for identicalDNA fragments 1A, 2A, 3A. In this case, it is possible to recognize thedegree of the reaction depending on the size of the spot, in addition toa digital inspection result to indicate whether or not the reactionoccurs, with the spots of the corresponding DNA fragment. It is possibleto obtain an analog inspection result for the specimen.

In the embodiment of the present invention, as shown in FIG. 15A, it ispossible to form a plurality of spots having different amounts per unitarea of amounts of a DNA fragment immobilized in the respective spots.In the example shown in FIG. 15A, when different species of DNA havingdifferent efficiencies of hybridization with a specimen are immobilizedfor the respective spots 1A to 3D with different types of DNA fragments,the amount per unit area of the amount of the DNA fragment immobilizedin each of the spots is changed.

Specifically, the spots are formed as follows. That is, the amount perunit area of the amount of the immobilized DNA fragment is large foreach of the spots 1A, 1D, 2B, 2C, 2D, 3A, 3D in which the DNA specieshaving a low efficiency of hybridization with the specimen isimmobilized. The amount per unit area of the amount of the immobilizedDNA fragment is intermediate for each of the intermediate spots 1B, 2A,3B. The amount per unit area of the amount of the immobilized DNAfragment is small for each of the spots 1C, 3C in which the DNA specieshaving a high efficiency of hybridization with the specimen isimmobilized. Therefore, it is possible to suppress the dispersion of theability to capture the specimen. Further, it is possible to suppress thedeterioration of the quantitative performance and the dispersion of theinspection result which would be otherwise caused by the difference indetection sensitivity between the spots.

In the embodiment of the present invention, as shown in FIG. 15B, it ispossible for an identical DNA fragment to form a plurality of spotshaving different amounts per unit area of amounts of the DNA fragmentimmobilized on the base plate respectively. The example shown in FIG.15B is illustrative of a state in which four spots A₁ to A₄ havingdifferent amounts per unit area of amounts of DNA fragments immobilizedon the base plate respectively are formed for the identical DNAfragments 1A, 2A, 3A. In this case, it is possible to recognize thedegree of the reaction depending on the amount of the DNA fragment perunit area, in addition to a digital inspection result to indicatewhether or not the reaction occurs, with the spots of the correspondingDNA fragment. It is possible to obtain an analog inspection result forthe specimen.

In the embodiment of the present invention, as shown in FIGS. 16 and 17,for example, a first layer spot 80A, which is formed on the base plate10, has a so-called doughnut-shaped configuration in which a peripheralportion 120 (see FIG. 17) is ridged, for example, by adjusting thedischarge power or the like of the micropipette 34. Further, after thespot 80A having the doughnut-shaped configuration is dried, a spot 80B,which contains a different DNA fragment and which has a substantiallycircular planar configuration, is formed on the spot 80A. Accordingly,the spots 80A, 80B, which contain the different samples respectively,can be formed at an identical spot formation position. In this case, itis possible to greatly reduce the arrangement area for the spot 80. Itis possible to miniaturize the DNA microarray 20 itself.

When the spots composed of different types of samples are formed at theidentical spot formation position, there is no limitation to thearrangement in which the formation position is allotted to theperipheral portion and the central portion of the spot as describedabove. However, when the effective spot areas are arrangedconcentrically as described above, an effect is obtained to reduce thedispersion of the shape between the different spots.

The DNA microarray as described above is preferably produced inaccordance with an ink-jet method. Especially, when different samplesare formed in an identical spot, or when spots of different samples arearranged in a contact manner, the production cannot be performed inaccordance with the conventional pin system, because of the problem ofpin contamination. However, the arrangement as described above can beefficiently realized with good accuracy by means of the ink-jet methodin which spots are formed in a non-contact manner.

It is a matter of course that the DNA microarray according to thepresent invention is not limited to the embodiments described above,which may be embodied in other various forms without deviating from thegist of the present invention.

INDUSTRIAL APPLICABILITY

As explained above, the following effects can be obtained in accordancewith the DNA microarray of the present invention.

(1) The inspection accuracy for the genetic analysis is improved, makingit possible to perform the quantitative evaluation.

(2) It is possible to achieve the high concentration of spots, and it ispossible to perform the detailed genetic analysis for a large amount ofsample at once.

(3) It is possible to recognize the degree of the reaction depending onthe amount of the immobilized DNA fragment, in addition to the digitalinspection result to indicate whether or not the reaction occurs. Thus,it is possible to obtain the analog inspection result for a specimen.

What is claimed is:
 1. A biochip comprising a large number of spotscontaining DNA arranged on a base plate, obtained by supplying, ontosaid base plate by means of an ink jet system, a plurality of types ofcapture solutions containing said DNA each of which is adapted tospecifically react with a specimen and provide information about astructure within the specimen, wherein: a plurality of said spots, whichhave different spot sizes, are formed on said base plate, wherein all ofsaid spots have uniform detection sensitivity.
 2. A biochip according toclaim 1, wherein said plurality of spots are formed from the samecapture solution.
 3. A biochip according to claim 2, wherein said spotscontaining said DNA are formed by a method using an ink-jet system, inwhich said capture solution is impacted onto said base plate after beingdischarged into the atmosphere, and wherein a force of the discharge iscontrolled electrically.
 4. A biochip according to claim 2, wherein saidspots containing said DNA are formed by a method using an ink-jetsystem, in which said capture solution is impacted onto said base plateafter being discharged into the atmosphere, and wherein the number oftimes of discharge at each spot and a force of the discharge areelectrically controlled, respectively.
 5. A biochip according to claim1, wherein said spots containing said DNA are formed by a method usingan ink-jet system, in which said capture solution is impacted onto saidbase plate after being discharged into the atmosphere, and wherein aforce of the discharge is controlled electrically.
 6. A biochipaccording to claim 1, wherein said spots containing said DNA are formedby a method using an ink-jet system, in which said capture solution isimpacted onto said base plate after being discharged into theatmosphere, and wherein the number of times of discharge at each spotand a force of the discharge are electrically controlled, respectively.7. A biochip comprising a large number of spots of DNA containing acapture material therein arranged on a base plate, obtained bysupplying, onto said base plate by means of an ink jet system, aplurality of types of capture solutions containing said DNA each ofwhich is adapted to specifically react with a specimen and provideinformation about a structure within the specimen, wherein: a pluralityof said spots are formed in which the concentration of the capturematerial in the capture solution varies from spot to spot, wherein allof said spots have uniform detection sensitivity.
 8. A biochip accordingto claim 7, wherein said plurality of spots are formed from the samecapture solution.
 9. A biochip according to claim 8, wherein said spotscontaining said DNA are formed by a method using an ink-jet system, inwhich said capture solution is impacted onto said base plate after beingdischarged into the atmosphere, and wherein a force of the discharge iscontrolled electrically.
 10. A biochip according to claim 8, whereinsaid spots containing said DNA are formed by a method using an ink-jetsystem, in which said capture solution is impacted onto said base plateafter being discharged into the atmosphere, and wherein the number oftimes of discharge at each spot and a force of the discharge areelectrically controlled, respectively.
 11. A biochip according to claim7, wherein said spots containing said DNA are formed by a method usingan inkjet system, in which said capture solution is impacted onto saidbase plate after being discharged into the atmosphere, and wherein aforce of the discharge is controlled electrically.
 12. A biochipaccording to claim 7, wherein said spots containing said DNA are formedby a method using an ink-jet system, in which said capture solution isimpacted onto said base plate after being discharged into theatmosphere, and wherein the number of times of discharge at each spotand a force of the discharge are electrically controlled, respectively.13. A biochip comprising a large number of spots of containing DNAtherein arranged on a base plate, obtained by supplying, onto said baseplate by means of an ink jet system, a plurality of types of capturesolutions containing said DNA each of which is adapted to specificallyreact with a specimen and provide information about a structure withinthe specimen, wherein: a plurality of said spots are formed in which theconcentration of said DNA in the capture solutions varies from spot tospot, wherein all of said spots have uniform detection sensitivity andsaid base plate comprises glass.